Polymerase Chain Reaction Technique Research Articles

Comparison of Gene Polymorphisms of ACE1 and ACE2 and the Level of Total ACE Activity in the Blood of Afghans and Iranians with COVID-19 and Its Relationship with Disease Severity.

Field evidence indicates differences in the rate and severity of COVID-19 infection among Afghans and Iranians, potentially influenced by individual genomic variances. Therefore, investigating the potential causes of these disparities holds significant clinical importance. This study aims to explore and compare variations in the genes encoding angiotensin-converting enzyme 1 (ACE1) and angiotensin-converting enzyme 2 (ACE2), along with total ACE activity levels in the blood of Afghans and Iranians with COVID-19, to assess any potential correlation with disease severity. In this case-control study, 124 Afghans and 124 Iranians with COVID-19 residing in Rafsanjan city, Iran, were examined. Blood samples were collected from all subjects, and serum was isolated for measuring total ACE activity using the kinetic method. DNA extraction was performed using the salting-out method, and gene polymorphisms of ACE1 and ACE2 were determined through polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism techniques. The DD genotype and D allele, as well as the GG genotype and G allele, were more prevalent among individuals with severe COVID-19 cases compared with those with mild symptoms, indicating an increased risk of severe infection. Although the Iranian group exhibited higher levels of these genetic components, along with longer hospital stays, intensive care unit admissions, and mortality rates than the Afghan group, the differences were not statistically significant. Furthermore, individuals with the DD genotype displayed double the total ACE activity levels compared with those with the II genotype, with the ID genotype falling in between. The presence of the DD genotype and D allele, as well as the GG genotype and G allele, likely serves as a significant risk factor for COVID-19 susceptibility, potentially heightening the risk of severe infection among Iranians compared with Afghans.

Clinical Utility of Broad-Range PCR Testing and Impact on Outcomes in Adults with Suspected Infection

Background: The development of broad-range polymerase chain reaction (BR-PCR) and next-generation sequencing techniques has significant implications for antimicrobial stewardship by increasing clinicians’ ability to provide a tailored antimicrobial regimen. We sought to explore the clinical utility of BR-PCR testing and its impact on antimicrobial treatment among an adult cohort in a large hospital system. Methods: We retrospectively evaluated samples that underwent BR-PCR testing between 2017 and 2021 and summarized their clinical characteristics and impact on antimicrobial therapy. We identified BR-PCR testing as having clinical utility if the results led to adjustment of antimicrobial choice or duration or to confirmation of the initial empiric regimen, while no clinical utility was assigned to results that were negative or clinically insignificant, unavailable due to loss to follow-up, or lacking clinical indication (treatment completed before the test results returned or conventional cultures revealed the causative pathogen). Results: Among 359 specimens, BR-PCR was positive for 107 (30%). Clinical utility was identified for 106 (29.5%) specimens, including 45 with negative BR-PCR results. The rates of clinical utility varied based on the type of sample tested, with the highest clinical utility associated with cranial samples (60%), followed by body fluid (56%) and endovascular (54%) samples, and the lowest with CSF (15%) and bone and joint (19%) samples. We also identified many BR-PCR tests that were not clinically indicated (23.4%). Conclusions: This study highlights the utility of BR-PCR testing to support antimicrobial stewardship initiatives. Further studies are needed to identify clinical scenarios in which it is appropriate to order BR-PCR testing and for a careful interpretation of negative BR-PCR results.

Ginkgo biloba extract alleviates deltamethrin-induced testicular injury by upregulating SKP2 and inhibiting Beclin1-independent autophagy

BackgroundMale infertility is a worldwide concern that is associated with a decline in sperm quality. Environmental pollutants such as deltamethrin (DM) have harmful effects on male reproductive organs. By maintaining intracellular redox homeostasis, ginkgo biloba extract (GBE) can alleviate male reproductive dysfunction. However, research on the mechanisms by which GBE alleviates reproductive toxicity induced by DM is limited. PurposeIn this study, we investigated whether GBE can alleviate DM-induced testicular and Sertoli cell reproductive toxicity by modulating SKP2 and Beclin1, thus providing a theoretical basis for the development of novel therapeutic approaches. Study DesignWe explored the role of GBE in mitigating DM-induced testicular damage, with a specific focus on the intricate involvement of ubiquitination and autophagy. MethodsAn experimental model was constructed using ICR male mice and the TM4 cell line. Tissue, cellular, and sperm morphological changes were observed through methods such as Hematoxylin and Eosin (H&E) staining, Periodate-Schiff (PAS) staining, ultrastructural observation, immunohistochemistry, and immunofluorescence. Enzyme and hormone levels were measured, and gene and protein levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting techniques. ResultsIn vivo experiments showed that DM exposure led to decreased sex hormone levels, increased seminiferous tubule diameter and impaired spermatogenesis. Meanwhile, DM exposure was found to decrease ubiquitination levels, leading to mitochondrial damage and further escalation of mitochondrial autophagy. Furthermore, in the DM-induced cell model, the upregulation of Beclin1 expression was associated with the inhibition of the ubiquitin‒proteasome system (UPS) and SKP2, thereby exacerbating autophagy. However, GBE has demonstrated notable efficacy in alleviating the reproductive toxicity induced by DM. ConclusionOur findings highlighted that SKP2 is a key regulator of Beclin1-independent autophagy and that GBE exerts therapeutic effects by upregulating SKP2 and inhibiting Beclin1 activation, which ameliorates autophagy and reduces DM-induced testicular damage.

Locally Manufactured Malaria RDTs for Plasmodium vivax: A Comparative Study of Kits Approved by National Medical Products Administration in China.

Early and accurate detection of Plasmodium vivax malaria cases is essential for preventing the re-establishment of malaria transmission in China, given the widespread distribution of Anopheles vectors that transmit vivax malaria. In this study, we aimed to compare the performance of three different commercial malaria rapid diagnostic test (RDT) kits for screening P. vivax in China. Microscopy was utilized to identify malaria parasites and to determine their parasite density levels. For the validation of microscopic findings and the genetic characterization of Plasmodium species, a nested polymerase chain reaction (PCR) technique was used. The Malaria RDT kits used in the present study encompassed products from BioPerfectus, which are designed for the detection of P. falciparum and other Plasmodium species, and kits from BlueCROSS and EGENS, tailored for the concurrent detection of P. falciparum and P. vivax. As a result, the overall agreement between malaria RDTs and nested PCR was almost perfect, with kappa values as follows: BioPerfectus (κ = 1.000), BlueCROSS (κ = 0.975, 95% CI 0.971-0.979), and EGENS (κ = 0.950, 95% CI 0.941-0.959). However, there was a statistically significant difference in the detection rate of P. vivax-positive samples from 94.5% (104/110) to 100% (P = 0.047). Furthermore, the agreement between the BlueCROSS and EGENS kits for detecting P. vivax-specific pLDH was almost perfect (P = 0.499, κ 0.973, 95% CI 0.968-0.978), while the detection rates were lower for samples with low parasite densities (BlueCROSS: P = 0.007; EGENS: P = 0.001). The detection capacity of the existing P. vivax-specific RDT kits in China needs to be further improved.

N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter.

The objective of this study was to identify the N6-methyladenosine (m6A)- circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA). The validation of circHECA m6A sites was performed using methylation immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique. The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA. The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

Clinical, biochemical, and genetic study of TACE/TNF-α/ACE signaling pathway in pediatric COVID-19 infection.

: Pediatric patients infected with coronavirus disease 2019 (COVID-19) have unique clinical characteri-stics. Tumor necrosis factor (TNF) is a proinflammatory cytokine that greatly contributes to tumor pathogenesis. : To describe the presenting characteristics of COVID-19 infection among pediatric patients, and investi-gate the possible role of the TNF-α signaling pathway. : This prospective case-control study included 50 Egyptian pediatric patients with COVID-19 and 50 healthy controls. Clinical, laboratory, and radiological as-sessments were performed. Serum TNF-alpha (TNF-α), TNF-α-converting enzyme (TACE), and angiotensin-con-verting enzyme 2 (ACE2) were measured using enzyme-linked immunosorbent assay. ACE (I/D) (rs4646994), ACE2 rs2285666, and TNF-α-308G/A single nucleotide poly-morphisms (SNPs) were performed using conventional polymerase chain reaction techniques with or without restriction fragment length polymorphism. : The median age was 1 year (interquartile range [IQR], 0.31-2.50 years) in the case group and 1.45 years (IQR, 1.00-3.00) in the control group. The main presenting symptoms were fever (92%), dry cough (74%), and dyspnea (72%). The lymphocytic count was normal in 14 patients (28%), decreased in 16 patients (32%), and in creased in 20 patients (40%) of the case group. Positive chest com-puted tomography finding of COVID-19 infection were demonstrated among 40% of patients using COVID-19 Re-porting and Data System categories (ground-glass op-acity with or without consolidations in the lungs). There were significant increased serum TACE and TNF-α with decreased ACE2 levels among cases versus controls (P< 0.001). The GG genotype and G allele of the TNF-α-308G/A SNP were significantly higher in patients than in controls (P<0.05 for both), with insignificant differences in genotype and allelic frequencies in the ACE (I/D) (rs4646994) and ACE2 rs2285666 SNPs. : The TNF signaling pathway was significantly activated in pediatric COVID-19 infection. Only the TNF-α-308G/A SNP was significantly associated with pediatric COVID-19 infection.

Preparation and Characterization of Essential oil from Lavandula spica Plant and its Antimicrobial Activity against Pseudomonas aeruginosa and Staphylococcus aureus

The biological properties of herbs and essential oils (EOs), such as their antibacterial, analgesic, anti-inflammatory, antioxidant, and anticancer characteristics, make them widely used in a variety of fields. This research aims to assess the antibacterial efficacy of lavender oil against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). The essential oil from Lavandula spica was extracted via water distillation and characterized by using Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR). Bacterial strains were isolated from burn wounds, confirmed by polymerase chain reaction technique, and were tested using disc diffusion method and Minimum Inhibitory Concentration (MIC) calculations. The study identified 28 components in the EO, with monoterpenes being the predominant. Out of 150 samples, 56 (45.52%) were positive for P. aeruginosa and 67 (54.47%) for S. aureus by using standard microbiological techniques including Gram staining, biochemical tests and Polymerase chain reaction technique. P. aeruginosa showed high resistance to cefazolin (100%) and levofloxacin (83.3%), while S. aureus was highly resistant to cefoxitin, piperacillin, amoxicillin/clavulanic acid, and ampicillin/sulbactam. The zone of inhibition and MIC for EO against P. aeruginosa were 9.910±0.866 and 2.376±0.352 while for S. aureus were 10.597±0.818 and 0.894±0.073 respectively with significance levels of p>0.05 and p<0.01. The study concluded that L. spica EO shows promising antimicrobial activity, particularly against Gram-positive bacteria suggesting its potential for further research and antimicrobial use.

Organophosphate and carbamate susceptibility profiling of Anopheles gambiae sl. across different ecosystems in southern Benin.

To overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile of Anopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial. Anopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3-5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species, kdr and Ace-1 mutations. Anopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereas Anopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested were Anopheles coluzzii. The frequencies of Ace-1R mutation in all Anopheles coluzzii populations tested were low (3-27%). Organophosphates (malathion and pirimiphos-methyl) have maintained their efficacy against Anopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates against Anopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area.

Grafting semi-wild tomato GZ-05 rootstocks improved cold tolerance via the signalling of melatonin and jasmonic acid

Tomato (Solanum lycopersicum) cultivation in the off-season is significantly hindered by cold stress; hence, utilising stress-resistant rootstocks in grafting is a critical solution. This study used 30 semi-wild tomato GZ-05 plants as rootstocks and Ailsa Craig (AC) tomatoes as scions. After cold stress, the scion tolerance index, leaf ion permeability, and other physiological and biological indicators were used to determine the most tolerant plants. To understand the molecular basis of GZ-05 rootstock cold stress resistance, RNA sequencing and reverse transcription polymerase chain reaction techniques were used to compare the varying genes expressed in the grafted AC/GZ-05 and self-grafted AC/AC plant scion leaves. The results indicated that genes associated with melatonin (MT) and jasmonic acid (JA) production and their signalling pathways were considerably altered. The initial MT and JA levels in the GZ-05-grafted plant scions were high, and when they were exposed to cold stress, the amount of active MT and JA in AC/GZ-05 heterologous grafts were great. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, we knocked out the MT synthesis gene (SlCOMT14) and JA synthesis gene (SlLoxD) and found that GZ-05 plant cold stress tolerance decreased. External tests were conducted to evaluate the GZ-05 SlCOMT14 and SlLoxD knockout lines. Source spraying with MT and methyl jasmonate showed that knockout strain cold stress tolerance could be recovered. SlICE1, a tomato cold stress tolerance transcription factor, was silenced, resulting in decreased tolerance to MT- and JA-induced cold stress. The MT/JA-inducer of C-repeat binding factor (CBF) expression 1-CBF pathway may be the mechanism by which the semi-wild tomato GZ-05 rootstock confers cold tolerance to plants. This study has uncovered the molecular mechanism by which grafting semi-wild tomato GZ-05 rootstocks increases plant cold tolerance, thereby laying the groundwork for the utilisation of Guizhou's native semi-wild tomato germplasm resources.

Insecticide resistance and its intensity in urban Anopheles arabiensis in Kisumu City, Western Kenya: Implications for malaria control in urban areas.

The rise of insecticide resistance poses a growing challenge to the effectiveness of vector control tools, particularly in rural areas. However, the urban setting has received comparatively less focus despite its significance in attracting rural to urban migration. Unplanned urbanization, often overlooked, exacerbates insecticide resistance as Anopheles mosquitoes adapt to the polluted environments of rapidly expanding cities. This study aimed to assess the insecticide susceptibility status of malaria vectors and identify potential underlying mechanisms across three distinct ecological settings characterized by differing levels of urbanization in Kisumu County, Kenya. The study was conducted in 2022-2023 in Kisumu County, western Kenya. Field-derived An. gambiae (s.l.) larvae collected from a long stretch of urban-to-rural continuum were phenotyped as either resistant or susceptible to six different insecticides using the World Health Organization (WHO) susceptibility test. Polymerase chain reaction (PCR) techniques were used to identify the species of the An. gambiae complex and screened for mutations at voltage-gated sodium channels (Vgsc-1014F, Vgsc-1014S, Vgsc-1575Y) and acetylcholinesterase (Ace1) target site mutation 119S. Metabolic enzyme activities (non-specific β-esterases and monooxygenases) were evaluated in mosquitoes not exposed to insecticides using microplate assays. Additionally, during larval sampling, a retrospective questionnaire survey was conducted to determine pesticide usage by the local inhabitants. Anopheles arabiensis dominated in urban (96.2%) and peri-urban (96.8%) areas, while An. gambiae (s.s.) was abundant in rural settings (82.7%). Urban mosquito populations showed high resistance intensity to deltamethrin (Mortality rate: 85.2% at 10x) and suspected resistance to Pirimiphos-methyl and bendiocarb while peri-urban and rural populations exhibited moderate resistance intensity to deltamethrin (mortality rate >98% at 10x). Preexposure of mosquitoes to a synergist piperonyl butoxide (PBO) significantly increased mortality rates: from 40.7% to 88.5% in urban, 51.9% to 90.3% in peri-urban, and 55.4% to 87.6% in rural populations for deltamethrin, and from 41.4% to 78.8% in urban, 43.7% to 90.7% in peri-urban, and 35% to 84.2% in rural populations for permethrin. In contrast, 100% mortality to chlorfenapyr and clothianidin was observed in all the populations tested. The prevalence of L1014F mutation was notably higher in urban An. arabiensis (0.22) unlike the peri-urban (0.11) and rural (0.14) populations while the L1014S mutation was more prevalent in rural An. gambiae (0.93). Additionally, urban An. arabiensis exhibited elevated levels of mixed function oxidases (0.8/mg protein) and non-specific esterases (2.12/mg protein) compared to peri-urban (0.57/mg protein and 1.5/mg protein, respectively) and rural populations (0.6/mg protein and 1.8/mg protein, respectively). Pyrethroids, apart from their use in public health through LLINs, were being highly used for agricultural purposes across all ecological settings (urban 38%, peri-urban 36% and rural 37%) followed by amidine group, with organophosphates, neonicotinoids and carbamates being of secondary importance. These findings show high resistance of An. arabiensis to insecticides commonly used for vector control, linked with increased levels of detoxification enzymes. The observed intensity of resistance underscores the pressing issue of insecticide resistance in urban areas, potentially compromising the effectiveness of vector control measures, especially pyrethroid-treated LLINs. Given the species' unique behavior and ecology compared to An. gambiae, tailored vector control strategies are needed to address this concern in urban settings.

Abstract 4137939: Mosaic loss of Y chromosome is associated with increased all-cause mortality in patients with ST-segment elevation myocardial infarction

Introduction: Mosaic loss of Y chromosome (LOY), a common subtype of somatic mosaicism, is characterized as loss of the entire Y chromosome in a mosaic manner and increases with age. Accumulating evidence has manifested the positive link between LOY and shorter life expectations in elderly men as well as common age-associated diseases, such as cancer and cardiovascular diseases. Experimental studies also demonstrated that hematopoietic LOY in mice induced cardiac fibrosis and led to increased mortality. However, little is known about LOY and prognosis of ST-segment elevation myocardial infarction (STEMI). Hypothesis: We hypothesized that LOY may be associated with lower survival rate of patients with STEMI. Aims: Investigating the impact of LOY in peripheral blood cells on prognosis of patients with STEMI. Methods: This study prospectively enrolled 928 males and 272 females undergoing primary percutaneous coronary intervention for STEMI. LOY was measured in 928 males using droplet digital polymerase chain reaction technique. Results: During a median follow-up of 3.99 years, 93 males (10.0%) and 45 females (16.5%) died. After adjusting for traditional cardiovascular risk factors, the risk of all-cause mortality in males with LOY ≥ 18% (the 90th percentile of LOY) increased to 2.45 (95% confidence intervals [CI]: 1.16–5.15, P = 0.018), 2.11 (95% CI: 1.11–4.01, P = 0.024), and 1.88 (95% CI: 1.02–3.45, P = 0.043) times during 1-, 2-, and 3-year follow-up, respectively, compared to males with LOY &lt; 18%. Among males without prior MI, those with LOY ≥ 18% had an increased risk of all-cause mortality compared to those with LOY &lt; 18% (adjusted hazard ratio 1.93, 95% CI: 1.01–3.67; P = 0.045); this was not observed for males with prior MI. Female patients had a 1.71-fold (95%CI: 1.04–2.81; P = 0.035) increased risk of all-cause mortality than males with LOY &lt; 18%, whereas the risk was similar to males with LOY ≥ 18%. Conclusion: LOY was associated with an increased risk of all-cause mortality in male STEMI patients, particularly in those without prior MI. Detecting LOY in STEMI patients could help further refine sex differences in prognosis and assist in identifying male patients with better prognoses.

Enhancement of Cognitive Function in Rats with Vascular Dementia Through Modulation of the Nrf2/GPx4 Signaling Pathway by High-Frequency Repetitive Transcranial Magnetic Stimulation

Repetitive transcranial magnetic stimulation (rTMS) represents a non-invasive therapeutic modality acknowledged for augmenting neurological function recovery following stroke. Nonetheless, uncertainties remain regarding its efficacy in promoting cognitive function recovery in patients diagnosed with vascular dementia (VD). In this study, VD was experimentally induced in a rat model utilizing the bilateral common carotid artery occlusion method. Following a recuperation period of seven days, rats were subjected to high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) at a frequency of 10 Hz. Cognitive function was assessed utilizing the Morris water maze test, and the levels of IL-6, TNF-α, SOD, GSH, MDA, and Fe2+ in cerebral tissue were quantitatively analyzed through enzyme-linked immunosorbent assay. Moreover, the gene and protein expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPx4) were meticulously investigated via quantitative polymerase chain reaction (qPCR) and Western blotting techniques. The use of HF-rTMS notably augmented cognitive function in rats with VD, concomitantly reducing neuroinflammation, oxidative stress, and ferroptosis within the brain. The group subjected to HF-rTMS demonstrated an increase in the levels of both proteins and genes associated with Nrf2 and GPx4, in comparison to the VD group. These results highlight the potential of HF-rTMS treatment in enhancing cognitive function in rats diagnosed with VD through the modulation of the Nrf2/GPx4 signaling pathway. This modulation, in turn, mitigates processes linked with neuroinflammation, oxidative stress, and ferroptosis. Nevertheless, additional studies are essential to comprehensively elucidate the underlying mechanisms and clinical implications of HF-rTMS treatment in the treatment of VD.

Cost-effectiveness and other considerations for different research techniques applied in ancient DNA analysis.

Ancient DNA (aDNA) analysis has developed rapidly since it first emerged in the 1980s, becoming an almost indispensable tool in anthropological and archaeological sciences. Earlier aDNA study was based on the polymerase chain reaction (PCR) technique, with which, unfortunately, modern DNA contamination and other authenticity issues were often incurred. These technical hurdles were soon overcome by application of advancements in the forms of the next generation sequencing (NGS) technique and others. However, since NGS requires money, time, and, in the case of large projects, manpower as well, genetic analysis of some ancient samples considered to be insignificant is commonly delayed or, in the worst cases, neglected entirely. We acknowledge that as a diagnostic tool in aDNA analysis, PCR is less accurate than NGS and more easily affected by modern DNA contamination; but it also has advantages, such as simplicity, time-saving, and greater ease of interpretation, among others. The role of PCR in aDNA analysis, then, should be reconsidered.

METTL3 alters AK3 RNA expression in an m6A-dependent manner to affect the proliferation and metastasis of hepatocellular carcinoma

In recent years, the significance of N6-methyladenosine (m6A) modification in the process of tumorigenesis has garnered substantial attention from researchers in the field. Among the myriad of factors involved, METTL3, which functions as an m6A methyltransferase, has emerged as a critical player in this context. This enzyme has been shown to participate actively in the regulation of RNA stability and expression across a diverse array of tumors. To achieve this, we employed quantitative polymerase chain reaction (qPCR) and Western blot analysis techniques to measure the expression levels of both METTL3 and AK3 in various hepatocellular carcinoma cell lines. We utilized small interfering RNA (siRNA) technology to inhibit the expression of METTL3, subsequently observing the resulting changes in AK3 expression. We analyzed the levels of m6A modification using the MeRIP-Seq method to obtain a comprehensive understanding of the underlying molecular interactions. Through MeRIP-Seq analysis, we discovered that METTL3 directly modulates the stability of AK3, thereby promoting its expression via m6A modification. This finding is pivotal as it underscores the regulatory role that METTL3 plays in AK3 RNA expression, facilitated through the M6A modification pathway.

Data normalization of plasma miRNA profiling from patients with COVID-19

When using the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) technique for quantitative assessment of microRNA (miRNA) expression, normalizing data using a stable endogenous gene is essential; however, no universally adequate reference gene exists. Therefore, in this study, we aimed to determine, via the RNA-Seq technique, the most adequate endogenous normalizer for the expression assessment of plasma miRNAs in patients with coronavirus disease 2019 (COVID-19). Two massive sequencing procedures were performed (a) to identify differentially expressed miRNAs between patients with COVID-19 and healthy volunteers (n = 12), and (b) to identify differentially expressed miRNAs between patients with severe COVID-19 and those with mild COVID-19 (n = 8). The endogenous normalizer candidates were selected according to the following criteria: (1) the miRNA must have a fold regulation = 1; (2) the miRNA must have a p-value > 0.990; and (3) the miRNAs that were discovered the longest ago should be selected. Four miRNAs (hsa-miR-34a-3p, hsa-miR-194-3p, hsa-miR-17-3p, and hsa-miR-205-3p) met all criteria and were selected for validation by RT-qPCR in a cohort of 125 patients. Of these, only hsa-miR-205-3p was eligible endogenous normalizers in the context of COVID-19 because their expression was stable between the compared groups.

Nuclear factor erythroid 2-related factor 2 alleviates lung endothelial cells injury by inhibition of ferroptosis.

In recent years, the survival rate of preterm infants has significantly improved due to the application of pulmonary surfactant (PS) and advancements in lung-protective mechanical ventilation strategies. However, this has been accompanied by an increased incidence of complications, particularly lung diseases triggered by elevated reactive oxygen species (ROS) induced by hyperoxia. The primary mechanism of hyperoxic lung injury (HLI) involves the excessive production of ROS within cells and the aggregation of inflammatory cells. Currently, no effective prevention or treatment methods are available. Ferroptosis, a newly identified form of cell death, is closely linked to ROS accumulation and is likely involved in HLI. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates both HLI and ferroptosis, and targeting Nrf2 to inhibit ferroptosis may represent a key therapeutic approach for treating HLI. This study aimed to investigate the involvement of ferroptosis in HLI and to elucidate the regulatory role of Nrf2. We employed the human pulmonary microvascular endothelial cell (HPMEC) model of hyperoxia exposure and corresponding intervention groups. Mitochondrial morphological alterations within HPMECs exposed to hyperoxia and various control groups were examined using transmission electron microscopy (TEM). Cell viability was assessed via the Cell Counting Kit-8 (CCK-8) assay, whereas intracellular ROS levels were quantified using the dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Furthermore, the expression levels of GPX4 and Nrf2 were analyzed through quantitative polymerase chain reaction (qPCR) and western blot techniques. Relative to the control group, the HPMECs subjected to hyperoxic conditions exhibited diminished viability, heightened ROS levels, decreased GPX4 expression, and increased Nrf2 expression. These cells also demonstrated mitochondrial morphological alterations characteristic of ferroptosis, including reduced mitochondrial cristae and shrinkage. The application of a ferroptosis inhibitor mitigated cellular damage, lipid peroxidation, and the morphological manifestations of mitochondrial ferroptosis, whereas Nrf2 inhibitor ML385 reversed this effect. Ferroptosis appears to contribute to the pathogenesis of HLI, with Nrf2 serving a protective function by mitigating ferroptosis.

Loganin inhibits the ROS-NLRP3-IL-1β axis by activating the NRF2/HO-1 pathway against osteoarthritis

Loganin (LOG), a bioactive compound derived from Cornus officinalis Siebold & Zucc, has been understudied in the context of osteoarthritis (OA) treatment. In this study, we induced an inflammatory response in chondrocytes using lipopolysaccharide (LPS) and subsequently treated these cells with LOG. We employed fluorescence analysis to quantify reactive oxygen species (ROS) levels and measured the expression of NLRP3 and nuclear factor erythropoietin-2-related factor 2 (NRF2) using real-time quantitative polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence (IF) techniques. Additionally, we developed an OA mouse model by performing medial meniscus destabilization (DMM) surgery and monitored disease progression through micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, safranin O and fast green (S&F) staining, and immunohistochemical (IHC) analysis. Our results indicate that LOG significantly reduced LPS-induced ROS levels in chondrocytes, inhibited the activation of the NLRP3 inflammasome, and enhanced NRF2/heme oxygenase 1 (HO-1) signaling. In vivo, LOG treatment mitigated cartilage degradation and osteophyte formation triggered by DMM surgery, decreased NLRP3 expression, and increased NRF2 expression. These findings suggest that LOG has a protective effect against OA, potentially delaying disease progression by inhibiting the ROS-NLRP3-IL-1β axis and activating the NRF2/HO-1 pathway.

HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-β/Smad signaling pathway.

The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-β/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-β/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.

A PCR-Based Approach for Early Diagnosis of Head and Neck Aspergillosis: A Pilot Study.

Aspergillosis is a fungal disease caused by the inhalation of fungal spores of the genus Aspergillus spp. This fungus mainly affects the lungs but can spread and infect the maxillofacial region through the bloodstream or inoculation of the fungus after extraction or endodontic treatment, especially in the upper posterior teeth. The disease has nonspecific clinical manifestations that hinder its early diagnosis. Although the Polymerase Chain Reaction (PCR) technique holds promise as a diagnostic tool for aspergillosis, anatomopathological analysis services do not routinely adopt this method. Therefore, the present study aimed to evaluate the applicability of PCR and standardise the techniques of preparation of biological samples for the detection of the three species: Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. Methods: Six samples of formalin-fixed, paraffin-embedded tissue (FFPE) with a histopathological diagnosis suggestive of aspergillosis were investigated using PCR. As a positive control for the PCR reaction, morphologically and genetically characterized cultures were used, with their sequences deposited at NCBI under accession codes MW837777 (A. fumigatus) and MW837779 (A. niger). The A. flavus culture used is reference RC 2053. Four of the six samples evaluated were positive for Aspergillus spp., of which one was co-infected with A. fumigatus and A. flavus species, while two others were positive only for A. flavus, and one sample was positive only for A. fumigatus. These findings suggest that PCR can be used as an auxiliary method for diagnosing aspergillosis. However, this was a pilot study, and expansion of the sample size and the evaluation of PCR in comparison with other diagnostic tests for aspergillosis are essential to determine the accuracy of the method.

Laparoscopic Salpingectomy Regulates The Expression of Endometrial Homeobox Genes (HOXA9, HOXA10, HOXA11, HOXD10) and HOX Transcript Antisense Intergenic RNA in Women with Communicating Hydrosalpinx: A Prospective Study.

This study aimed to determine the alteration of endometrial expression levels of HOXA9/HOXA10/ HOXA11/HOXD10 genes and HOX transcript antisense intergenic RNA (HOTAIR) in mid-luteal phase endometrium in patients with hydrosalpinx before and after salpingectomy. In this prospective study, 14 infertile women with unilateral hydrosalpinx who were scheduled for laparoscopic salpingectomy were evaluated. The presence of hydrosalpinx was confirmed by hysterosalpingography or transvaginal 2D-ultrasonography. All patients had normal hormonal profiles, body mass index, and regular menstrual cycles identified by mid-luteal serum progesterone. Fourteen healthy fertile age-matched women with a successful pregnancy history were considered the control group. Mid-luteal-phase endometrial biopsies were performed at the time of surgery and during the mid-luteal phase of the fourth treatment cycle by Pipelle. After tissue collection, RNA extraction and cDNA synthesis were performed. Real-time polymerase chain reaction (PCR) technique was used for quantitative gene expression of HOXA9/HOXA10/ HOXA11/ HOXD10 and lnc HOTAIR. The endometrial expression of HOXA9 (P<0.001), HOXA10 (P=0.001), HOXA11 (P=0.003), and HOXD10 (P=0.004) were significantly lower in the patients with hydrosalpinx compared to the controls. After salpingectomy, we observed a significant increase in the endometrial HOXA9 (P=0.006) and HOXA10 (P=0.023) mRNA expression levels compared to before salpingectomy samples. Similarly, a significant upregulation in endometrial HOXA11 (P= 0.013) and HOXA10 mRNA expression levels (P=0.012) were detected in postoperative samples compared to preoperative tissue. Moreover, the lnc HOTAIR was significantly higher in the endometrium-induced hydrosalpinx fluid than in controls (P=0.020), which had a 2.89-fold decrease following salpingectomy (P=0.010). Elevated endometrial lncRNA HOTAIR may disrupt the expression of endometrial receptivity HOX genes in women with hydrosalpinx. However, our results failed to show a significant inverse correlation between HOTAIR and HOX genes due to limited sample size. Further studies with larger sample sizes are required to investigate HOTAIR inverse co-expression with HOX genes in these subjects.